The results of nitric oxide (NO) infusions in normal volunteers and NO infusions and inhalation in experimental animals confirms that NO can be transported as a hormone and thus has the potential to be a pharmacological agent (i.e., a drug). We believe that the lack of vascular effects in our sickle cell patients is due to the presence of circulating hemoglobin and that this contributes to the pathophysiology of this and other chronic and acute hemolytic syndromes, especially the pulmonary hypertension complications which we have found to be severe and of high frequency in older patients. We have analyzed the effects of methemoglobin formation on blood pressure and other cardiovascular parameters in dogs to see if the ferric species of hemoglobin-which could be pharmacologically effected- is safe as has been assumed. To our suprise we find that infusions of methemoglobin lead to prolonged increases in blood pressure and systemic resitance and believe that the mechanisms relates to reduction of methemoglobin to ferrous hemoglobin by plasma ascorbate and the destruction of plasma or cellular NO and nitrite by this species. We also find that NO-bioactivity destruction appears to occur in tissues to a much greater extent than in the vascular bed. An alternative approach to mitigate the effects of cell-free hemoglobin is the infusion of haptoglobin or other agents which may bind hemoglobin or some of its degradation products. Studies of these pathways, in animals and possibly patients, are now being planned. We have also been fortunate in being able to use the CC animal facility and test inhaled nitrite and nitrate ions in hypoxic dogs to see if administering NO bioactivity in this way would be therapeutically possible. We have found strong effects of nitrite inhalation on both pulmonary and systemic parameters but the effects of nitrate (perhaps as expected) are only seen intermittently, suggesting that nitrate in the salivary fluid is reduced to nitrite and then systemic and pulmonary exposure give NO effects due to nitrite reduction processes. We have recently resumed our work on platelet and blood clotting inhibition by nitrite and are studying the effects of changes in ambient oxygen levels on the processes in rodent models using the TEG methodologies. We have also measured the effects of red cell reduction of plasma nitrite on platelet signaling and have confirmed that VASP phosphorylation occurs with the generation of NO. We have now used this parameter to characterize other aspects of this overall process. Our third major new project has been to measure nitrate and nitrite levels in various rodent organs. Our data on levels in organs with NOx dietary modification has suggested that active transport of nitrate may occur in several organs. We are now testing if sialin or another protein could account for these findings. However, perhaps our most surprising result was our finding of very high nitrate levels in muscle tissue. These levels are much higher than in blood and not only suggest active transport into muscle but also production of NO and oxidation to nitrate in the tissue itself. Our preliminary results suggest that it is the latter process, with production via NOS1 (nNOS) and oxidation due to reaction with oxymyoglobin, that most likely explain these observations. Most excitingly we have found that exercise, which can increase blood flow 10- or 20-fold, causes marked fall in nitrate levels and transient increases in muscle nitrite levels. We postulate that muscle tissue has a specific mechanism for nitrate reduction, possibly using xanthine oxidoreductase and this process may explain the long mysterious mechanism by which a muscle-produced agent caused marked changes in organ blood flow.